Abstract 570: Developing a novel adaptor CAR-T cell platform based on the recognition of the P329G Fc mutation in therapeutic IgG1 antibodies for adoptive T cell therapy
Authors: Diana Darowski, Mohamed-Reda Benmebarek, Christian Jost, Kay Stubenrauch, Uwe Wessels, Joerg Benz, Anne Freimoser-Grundschober, Ekkehard Moessner, Pablo Umana, Sebastian Kobold, Christian Klein
Journal: Cancer Research (2022)

Abstract Chimeric antigen receptor (CAR) T cells have shown promising results for the treatment of blood cancers and various CAR-T cell approaches are in development for use in different tumor indications. Universal or modular CARs do not directly recognize the tumor target antigen, but bind via an adaptor molecule to their respective tumor target. We describe the P329G-CAR-T platform as a novel modular CAR-T cell platform that recognizes the P329G mutation in the Fc portion of IgG1 antibodies, a mutation frequently applied to abolish the Fc immune effector function of therapeutic antibodies. In contrast to other adaptor CAR-T cell platforms this approach does not rely on haptens or artificial tags fused to the targeting antibody.The crystal structure analysis of the anti-P329G Fab fragment in complex with a P329G-Fc portion showed that the Fab fragment recognizes the Fc mutation with 1:1 binding stoichiometry. Surface plasmon resonance analysis determined the equilibrium binding affinity of the P329G antibody to the P329G Fc-portion to be 15 nM. Cell assays using Jurkat-NFAT reporter cell lines and primary T cells transduced with the P329G-CAR showed specific recruitment of P329G CAR-T cells by P329G-containing antibodies, and potent and dose dependent tumor cell killing accompanied by IFNg release and subsequent T cell activation for several unrelated tumor antigens including CD20, CD33, HER2, FOLR1 and mesothelin. Notably, P329G-CAR-T cell killing activity was comparable to the activity of the respective direct scFv-based CAR-T cells, both in terms of kinetics and absolute killing potency. Finally, comparable activity was determined in comparison to CD16 extracellular domain (ECD)-based CAR-T cells engaging the CAR-T cells via the Fc-CD16-ECD interaction. In summary, P329G-CAR-T cells mediate potent tumor cell killing in combination with various tumor targeting antibodies as adaptor molecules. The combination with tumor targeting antibodies enables control of CAR-T activity by adjusting the dose and schedule of the respective antibody adaptor molecule. Importantly, different from CD16-ECD-based CAR-T cells, P329G-CAR-T cells cannot be engaged by endogenous immunoglobulins. In vivo studies to investigate efficacy and safety of P329G CAR-T cells are currently being completed and will be reported. Citation Format: Diana Darowski, Mohamed-Reda Benmebarek, Christian Jost, Kay Stubenrauch, Uwe Wessels, Joerg Benz, Anne Freimoser-Grundschober, Ekkehard Moessner, Pablo Umana, Sebastian Kobold, Christian Klein. Developing a novel adaptor CAR-T cell platform based on the recognition of the P329G Fc mutation in therapeutic IgG1 antibodies for adoptive T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 570.